Large randomized gene libraries are an invaluable tool for the up-to-date protein engineering. Several methods have been developed for site-directed mutagenesis sharing the common aims to gain large high quality mutant libraries with as easy and fast protocol as possible. The main factors affecting the randomized gene library quality are the unintended biases in the gene diversity, the frequency of frame-shifts in the DNA sequence of the translated regions and the amount of wild type template molecules present in the final product. In the site-directed mutagenesis techniques a synthetic appropriately randomized oligonucleotide is incorporated into the target gene either by de novo gene assembly or by using wild type gene template. In the latter option, mostly used in research laboratories, the mutagenizing oligo is incorporated by primer extension in moderate temperature or PCR based methods using the wild type gene as the template.
International patent publication WO 2008/067035 discloses a technique called Unrestricted Mutagenesis and Cloning (URMAC) for mutagenizing large linear or circular nucleic acids. The technique involves a series of two PCR and three ligation steps with an addition optional enrichment PCR reaction.
One of the early primer extension methods uses uracil-containing single-stranded DNA (ss(U)DNA) template. No phenotypic selection is needed as the nascently synthesized mutant strand contains no uracil and is thus favoured in bacterial propagation resulting in reported 50% mutagenesis efficiency. This method, termed Kunkel mutagenesis, has subsequently been modified to be suitable for ds(U)DNA template by additional nitrocellulose filtering steps of alkali denatured template DNA, utilizing another oligo to destroy a unique restriction enzyme site for template sequence removal and by digesting remaining unmutated methylated ds(U)DNA template with DpnI. However, the ds(U)DNA template strategies compromise ease of template preparation with smaller library size as the primer extension with dsDNA compared to ssDNA is less efficient. Kunkel mutagenesis has even been successfully used to incorporate several mutagenesis oligos simultaneously into ss(U)DNA template. This strategy requires that the template DNA sequence is modified to contain stop codons at the sites of mutagenesis to prevent the translation of the wild type protein, as high proportion of the wild type template in the final product is the major drawback of the method.
There is a need in the art for an efficient, low-background mutagenesis method, in particular for creating large randomized gene libraries, wherein no template modifications are needed and which yields in ample supplies of transformable DNA circumventing the well-known problem that transformation of the DNA to host cells is the biggest bottleneck in the gene library production line.